There are five major types of dopamine receptors, classified into two primary categories, D1-like and D2-like. The D1-like family consists of the D1 and D5 dopamine receptors, whereas, D2-like family consists of the D2, D3, and D4 dopamine receptors. The D1 dopamine receptor (D1R), in particular, may be linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition. Agents that enhance D1R signaling in the prefrontal cortex may be useful in the treatment of cognitive decline in schizophrenia, Alzheimers disease and other age-related disorders. Unfortunately, orthosteric D1R agonists have proven problematic as they frequently induce hypotension and exhibit a narrow therapeutic window. An alternative approach is to potentiate D1R activity using allosteric modulation. Hypothetically, a positive allosteric modulator of the D1R may exhibit highly selective actions and exhibit a larger therapeutic window. In order to identify allosteric ligands of the D1R, we have implemented a high throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library. A D1R-G15 calcium mobilization assay was used to identify library compounds that potentiated an EC-20 concentration of dopamine. Initial hit compounds were characterized using endogenous G-protein (cAMP stimulation) and beta-arrestin (recruitment) signaling pathways, and parental cell lines were used as negative controls. A pool of compounds that exhibited positive allosteric modulation (PAM) of the D1R were identified and further counter-screened against other dopamine receptor subtypes to establish subtype selectivity. Two structurally diverse compounds, MLS6585 and MLS1082, with PAM activity at the D1R were selected as lead compounds. These compounds potentiate both G-protein and beta-arrestin-mediated D1R signaling, increasing the potency of dopamine by 5-10 fold for stimulating cAMP accumulation and 4-8 fold and for beta-arrestin recruitment. Both compounds also increased the maximum dopamine-stimulated responses by 25-30%. Neither compound displayed any intrinsic agonist activity in either assay. Further, the two compounds displayed minimal to no ability to inhibit radioligand binding to the orthosteric-binding site of the D1R. Both compounds potentiated the agonist activity of dopamine, as well as other full and partial agonists, in a dose-dependent manner with EC-50 values in the micromolar range. Importantly, they showed no ability to potentiate forskolin-stimulated cAMP accumulation, or beta-arrestin recruitment by other receptor subtypes. MLS1082 and MLS6585 may serve as important scaffolds for the future development of optimized D1R PAMs for in vivo use and the discovery of therapeutic lead compounds. We are currently engaged in a chemical optimization campaign to synthesize analogs around these scaffolds to both interrogate structure-activity relationships and optimize compounds for in vivo testing. Despite its clinical importance in the treatment of a number of neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, there are few compounds, either agonists or antagonists that are highly selective for the D2 dopamine receptor (D2R). Most compounds with activity at the D2R also exhibit significant affinity for the D3 or D4 dopamine receptors (D3R or D4R), or other G protein-coupled receptors (GPCRs). Development of D2R-selective ligands has proven especially problematic due to the high structural similarities of the orthosteric binding sites of the D2-like receptors (D2R, D3R and D4R). Importantly, highly selective D2R antagonists could represent improved therapeutics for treating the positive symptoms of schizophrenia with potentially fewer side effects due to off-target activities. Thus, in order to identify selective antagonists of the D2R, we have implemented a high-throughput screening (HTS) campaign to interrogate over 400,000 unique compounds in the Molecular Libraries Screening Center Network library. Not surprisingly, most of the compounds that were identified as antagonists of the D2R were also antagonists of the closely related D3R. However, a small pool of antagonist compounds that exhibited significant D2R>D3R selectivity was identified. These compounds were characterized using orthogonal D2R and D3R functional assays, including G protein-mediated assays and beta-arrestin recruitment, as well as radioligand binding assays. One compound, MLS1946, was found to exhibit high affinity for the D2R (Ki 100 nM) without measurable affinity for the D3R (Ki >40 micromolar). This compound was further characterized using a number of D2R and D3R signaling assays and found to exert no activity at the D3R, while exhibiting potent antagonism of the D2R. Follow-up studies were performed using an analog of the hit compound and a functional screen (beta-arrestin recruitment) of a panel of 168 different GPCRs. We found that the analog was completely devoid of agonist activity within this panel of GPCRs. Moreover, at the dose tested, the compound was found to completely antagonize dopamine stimulation of the D2R while exhibiting partial antagonism of the D4R. Surprisingly, the compound was devoid of significant antagonist activity at the remaining 166 GPCRs (including the D3R). These results suggest that the MLS1946 scaffold displays a highly D2R-selective antagonist profile. We are currently engaged in a chemical optimization campaign to synthesize analogs around this scaffold to both interrogate structure-activity relationships and optimize compounds for in vivo testing. It is anticipated that further development of the MLS1946 scaffold may lead to an optimized D2R-selective antagonist with drug-like properties for the treatment and understanding of dopamine-related pathologies. The D3R is enriched in the ventral striatum, which is associated with control of mood and emotion, while the D2R is enriched in the dorsal striatum, which is associated with the control of movement. Therefore, a D3R-selective antagonist may be useful for treating schizophrenia as it might attenuate psychotic symptoms without the motor side effects frequently induced by current antipsychotics, which are all D2R-preferring antagonists. Studies also suggest that D3R-preferring antagonists attenuate drug-seeking behaviors and relapse without causing the motor deficits associated with non-selective antagonists. In an effort to discover highly selective allosteric antagonists for the D3R, our lab has employed a high throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library. The library was initially screened using a D3R-mediated beta-arrestin recruitment assay. Confirmation and counter-screens were performed to obtain an initial assessment of D3R selectivity and mechanisms of action. 57 potential negative allosteric modulators (NAMs) were identified. Further triage of these compounds based on D3R vs. D2R selectivity in the beta-arrestin recruitment assay yielded 3 lead NAMs, the most potent of which was selected for further characterization. The compound was found to be completely D3R vs. D2R selective in a BRET-based beta-arrestin recruitment assay. Further Schild-type functional assays indicated that this compound acts as a noncompetitive antagonist of the D3R. In addition, a radioligand binding screen of this compound on 50 closely related GPCRs indicates this it shows limited to no activity at other receptors. Taken together, the data indicate that the compound is a highly selective negative allosteric modulator of the D3R. We will use medicinal chemistry efforts in order to further improve its potency, and we ultimately hope that this compound or its analogs will prove useful as in vitro and in vivo pharmacological tools or leads for therapeutic drugs.